Methods and reagents for cleaving and deprotecting oligonucleotides

ABSTRACT

Disclosed are reagents of methods for cleaving and deprotecting insolubilized and protected synthetic oligonucleotides. In a particularly preferred embodiment, the reagent comprises methylamine and t-butylamine.

RELATED APPLICATION

The present application is a divisional application of U.S. applicationSer. No. 07/873,915, filed Apr. 24, 1992, now U.S. Pat. No. 5,348,868.

The present application is related to U.S. Ser. No.. 07/873,330 (BeckmanDocket No. 128D-1007) for "Protecting Groups Useful In OligonucleotideSynthesis" by Parameswara Meda Reddy and Naeem Botros Hanna, which isfiled simultaneously herewith. The related application is incorporatedfully herein by reference.

FIELD OF INVENTION

The present invention relates generally to the synthesis ofoligonucleotides and more particularly to methods and reagents forcleaving oligonucleotides from solid supports, and removing protectinggroups from oligonucleotides.

BACKGROUND OF THE INVENTION

Deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") are long,threadlike macromolecules, DNA comprising a chain ofdeoxyribonucleotides, and RNA comprising a chain of ribonucleotides. Anucleotide consists of a nucleoside and one or more phosphate groups; anucleoside consists of a nitrogenous base linked to a pentose sugar.Typically, the phosphate group is attached to the fifth-carbon ("C-5")hydroxyl group ("OH") of the pentose sugar; however, it can also beattached to the third-carbon hydroxyl group ("C-3 OH"). In a molecule ofDNA, the pentose sugar is deoxyribose, while in a molecule of RNA, thepentose sugar is ribose. The nitrogenous bases in DNA are adenine ("A"),cytosine ("C"), guanine ("G"), and thymine ("T"). These bases are thesame for RNA, except that uracil ("U") replaces thymine. Accordingly,the major nucleosides of DNA, collectively referred to as"deoxynucleosides" are as follows: deoxyadenosine ("dA"); deoxycytidine("dC"); deoxyguanosine ("dG"); and thymidine ("T"). The correspondingribonucleosides are designated as "A"; "C"; "G"; and "U". (Byconvention, and because there is no corresponding thymidineribonucleoside, deoxythymidine is typically designated as "T"; forconsistency purposes, however, thymidine will be designated as "dT"throughout this disclosure).

The sequence of the nitrogenous bases of the DNA or RNA molecule encodesthe genetic information contained in the molecule. The sugar andphosphate groups of a DNA or RNA molecule perform a structural role,forming the backbone of the molecule. Specifically, the sugar moiety ofeach nucleotide is linked to the sugar moiety of the adjacent nucleotidesuch that the 3'-hydroxyl of the pentose sugar of one nucleotide islinked to the 5'-hydroxyl of the pentose sugar of the adjacentnucleotide. The linkage between the two pentose sugars is typically viaa phosphodiester bond. Based upon this linkage protocol, one end("terminus") of the nucleotide chain has a 5'-terminus (e.g. hydroxyl,triphosphate, etc.), and the other end has a 3'-hydroxyl group. Byconvention, the base sequence of a nucleotide chain is written in a 5'to 3' direction, i.e., 5'-ATCG-3' or simply ATCG

DNA and RNA are produced internally by living animals; however, DNA andRNA can be chemically synthesized such that synthetic strands of DNA andRNA can be rapidly and efficiently produced. These strands are typicallyreferred to as "synthetic oligonucleotides" or "oligonucleotides." Awidely utilized chemical procedure for the synthesis of oligonucleotidesis referred to as the "phosphoramidite methodology." See, e.g., U.S.Pat. No. 4,415,732; McBride, L. and Caruthers, M. Tetrahedron Letters,24:245-248 (1983); and Sinha, N. et al. Nucleic Acid Res. 12:4539-4557(1984), which are all incorporated herein by reference. Commerciallyavailable oligonucleotide synthesizers based upon the phosphoramiditemethodology include, e.g., the Biosearch 8750™ and ABI 380B™, 392™ and394™ DNA synthesizers.

The importance of chemically synthesized oligonucleotides is principallydue to the wide variety of applications to which oligonucleotides can bedirected. For example, oligonucleotides can be utilized in biologicalstudies involving genetic engineering, recombinant DNA techniques,antisense DNA, detection of genomic DNA, probing DNA and RNA fromvarious systems, detection of protein-DNA complexes, detection of sitedirected mutagenesis, primers for DNA and RNA synthesis, primers foramplification techniques such as the polymerase chain reaction, ligasechain reaction, etc, templates, linkers, and molecular interactionstudies.

The primary structures of DNA and RNA molecules can be depicted asfollows: ##STR1##

The key step in nucleic acid synthesis is the specific and sequentialformation of internucleotide phosphate linkages between a 5'-OH group ofone nucleotide and a 3'-OH group of another nucleotide. Accordingly, inthe typical synthesis of oligonucleotides, the phosphate group of an"incoming" nucleotide is combined with the 5'-OH group of anothernucleotide (i.e. the 5'-OH group is "phosphorylated" or"phosphitylated"). These groups must be capable of activelyparticipating in the synthesis of the oligonucleotides. Thus, the 5'-OHgroups are modified (typically with a dimethoxy trityl ("DMT") group)such that an investigator can introduce two such nucleotides into areaction chamber and adjust the conditions therein so that the twonucleotides are properly combined; by a series of successive suchadditions, a growing oligonucleotide having a defined sequence can beaccurately generated.

The four bases of the nucleosides, adenine, thymine (uracil in the caseof RNA), guanine and cytosine, include moieties which are chemicallyreactive (e.g., exocyclic amino groups). These groups must be"temporarily" protected, i.e. the protecting groups must be capable ofblocking any reactive sites on the base until after the oligonucleotidesynthesis is completed; after such synthesis is completed, these groupsmust also be capable of being removed from the bases such that thebiological activity of the oligonucleotide is not affected. Withoutprotecting the amino groups of dA, dC, dG (or the correspondingribonucleotides in the case of RNA synthesis), undesirable and/or lessuseful material will be synthesized. Thymine (T), which does not have anamino group, does not typically require a protecting group. Thetraditional amino protecting group for dA is a benzoyl group ("bz"); fordC, a bz group or an isobutyryl group ("ibu"); and for dG, ibu. It isconventional to indicate which protecting group is being utilized asfollows: dA^(bz), indicating that a benzoyl protecting group is beingused to protect the adenine exocyclic amino group.

In order to maintain an additional degree of control overoligonucleotide synthesis, it is necessary to initiate the synthesis ofthe oligonucleotides from an insolubilized starting point. I.e., theoligonucleotide is synthesized while "tethered" to an appropriate solidsupport. This protocol increases the speed and convenience of thesynthesis as compared to solution-based oligonucleotide synthesis.Accordingly, the synthesis of oligonucleotides requires, as a final stepor steps, the removal of protecting groups from the oligonucleotide("deprotection") and release ("cleavage") of the oligonucleotide from asolid support material--such steps are required in order to generate abiologically useful oligonucleotide.

"Deprotection" is defined as the process and the time necessary forremoval of protecting groups incorporated into the nucleotides;"cleavage" is defined as the process and the time necessary for removalof the synthesized oligonucleotide from a solid support material ontowhich the nucleotides have been attached.

Deprotection of dG is the rate limiting step for oligonucleotidedeprotection. By convention, those in the art will focus on the timenecessary to deprotect deoxyguanosine incorporated into theoligonucleotide. With respect to side-product formation (i.e. theformation of undesirable materials), deoxycytidine is particularlyvulnerable thereto. Accordingly, it is generally considered useful tomonitor side-product formation relative to dC in an effort to ensurethat side-product formation is avoided or minimized.

Deprotection and cleavage are typically accomplished utilizing ammonia.Because of the widespread use of ammonia as a deprotection and cleavagereagent, it is conventional to compare other such reagents with ammoniain terms of the times necessary for deprotection and cleavage.Deprotection and cleavage by means of ammonia can require several daysat room temperature, or between about 6 and 17 hours at 55° C.Typically, cleavage occurs within about 1 hour at room temperature, theremainder of the time being devoted to deprotection. The synthesis of atypical oligonucleotide (i.e. about 20-25 nucleotides in length) can besynthesized in about 2 hours; approximately six minutes is required toadd one nucleotide to the insolubilized and growing nucleotide chainusing most commercially available synthesizers. Relative to the timerequired to synthesize an oligonucleotide, the amount of time necessaryto deprotect and cleave the oligonucleotide using ammonia can beexcessive.

The use of different protection groups allows for a shortening of thetime required for deprotection and cleavage. It has been reported thatdimethylformamidine ("DMF") is a useful protection group for dG and dA.See Vu, Hugnh et al. "Fast oligonucleotide deprotection phosphoramiditechemistry for DNA synthesis". Tetrahedron Letters, 31:7269-7272 (1990)which is incorporated herein by reference. As described,oligonucleotides including such protection groups require 1 hour at 55°C. in aqueous ammonia, or 8 hours at room temperature in aqueousammonia, for deprotection and cleavage. It has also been reported thatphenoxyacetyl ("PAC") is a useful protection group for dG and dA. See,EPO Published Application 0241363 A1, "Derives de nucleotides et leurutilization pour 1a synthese d'oligonucleotides", Molko, Didier et al(1987) which is incorporated herein by reference. As described,oligonucleotides including this protection group require between 2 to 8hours at room temperature in aqueous ammonia for deprotection andcleavage.

An alternative approach to reduce the time required for deprotection andcleavage is to use other deprotection and cleavage reagents alone or inconjunction with ammonia. It has been reported that a reagent comprisinga lower alkyl alcohol, water and a non-nucleophilic hindered alkylaminecontaining from 3 to 6 carbon atoms provides for cleavage within 1-2hours at room temperature, followed by deprotection in about 20 to 60minutes at about 80° C. to about 90° C. See EPO Published Application0323152 A2, "Methods of Synthesizing Oligonucleotides", Woo, Sam Lee, etal (1989) (hereafter "Woo") which is incorporated herein by reference.Treatment of oligonucleotides with a 1:1 mixture (v/v) ofn-butylamine/methanol for deprotection and cleavage has been described.See Weber, H. and Khorana, H. G. "CIV. Total synthesis of the structuralgene for an alanine transfer ribonucleic acid from yeast. Chemicalsynthesis of an icosa-deoxynucleotide corresponding to the nucleotidesequence 21 to 40." J. Mol. Biol. 72:219-249 (1972) (hereinafter"Weber") which is incorporated herein by reference. However, thisreagent is reported to have led to the formation of deoxycytidineside-products. Therefore, and as further described, oligonucleotides notcontaining cytidine where treated with a 1:1 mixture (v/v) ofn-butylamine and methanol at room temperature for 2 days, and foroligonucleotides containing cytidine, treatment first included ammoniaat room temperature for 2 days, (to avoid the formation of deoxycytidineside-products) followed by a 1:1 mixture (v/v) of n-butylamine andmethanol at room temperature for 2 days.

The following is a brief summary of the described times necessary fordeprotection and cleavage for the foregoing protecting groups andreagents:

    ______________________________________                                        DEPROTECTION/CLEAVAGE TIMES                                                   REAGENT      TIME (HOURS)    TEMP (°C.)                                ______________________________________                                        Ammonia      6-17            55                                               Ammonia      >24             R.T.                                             DMF/Ammonia  2               55                                               DMF/Ammonia  8               R.T.                                             PAC/Ammonia  8               R.T.                                             Woo*         1-3             R.T.; 80/90                                      Weber**      2 days          R.T.                                             Weber***     4 days          R.T.                                             ______________________________________                                         *mixture of lower alkyl alcohol, water, nonnucleophilic hindered              alkylamine containing from 3-6 carbon atoms; 1-2 hours at R.T. (cleavage)     and 20-60 minutes at 80-90° C. (deprotection)                          **nonC containing oligonucleotides: 1:1 mixture (v/v)                         nbutylamine/methanol.                                                         ***oligonucleotide including C: ammonia (2 days) followed by                  nbutylamine/methanol (2 days).                                                R.T. = room temperature                                                  

While certain of the foregoing reported reaction times for cleavage anddeprotection are less than that of ammonia alone, it is evident thatrelative to the time necessary to synthesize an oligonucleotide, muchtime is devoted to deprotection and cleavage.

What is needed, given the rapidity in which oligonucleotides can besynthesized, is a reagent that can both rapidly remove the variety ofavailable protection groups, and cleave the oligonucleotide from thesupport, with minimal to no side-product formation. From a practicalperspective, it is preferred that such a reagent be useful acrossvarious temperatures, such that, in conjunction with the step ofheating, the rapidity at which such a reagent can both deprotect andcleave an oligonucleotide is increased.

SUMMARY OF THE INVENTION

The present invention satisfies at least this need. In accordance withthe invention disclosed herein, a reagent useful in the deprotection andcleavage of synthetically produced oligonucleotides is provided. Thereagent comprises at least one straight chain alkylamine having frombetween 1 to about 10 carbon atoms. Preferably, the reagent additionallycomprises at least one transamination suppression reagent, witht-butylamine being a particularly preferred transamination suppressionreagent. The reagent can further comprise ammonia.

Preferably, the alkylamine of the reagent has from between 1 to about 6carbon atoms, more preferably between 1 to about 3 carbon atoms, andmost preferably, 1 carbon atom. Accordingly, a most preferred reagentcomprises methylamine. Advantageously, deprotection and cleavage ofoligonucleotides can take place at room temperature in less than about90 minutes; however, higher temperatures may also be utilized to achievefaster deprotection of cleavage.

In accordance with the present invention, methodologies for cleaving aprotected oligonucleotide, removing the protecting groups from a solubleoligonucleotide, and deprotecting an insolubilized oligonucleotide, inconjunction with the disclosed reagent, are possible.

These and other advantages will be made apparent as the disclosureproceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are intended to be used for purposes ofillumination of the Detailed Description of Preferred Embodiments:

FIG. 1 is a graphic representation of the time for cleavage ofinsolubilized, heterogeneous 21-mers, using various reagents;

FIG. 2 is a melting point analysis of a 21-mer (and its complement)subjected to methylamine/t-butylamine as a cleavage/deprotectionreagent, and ammonia as a cleavage/deprotection reagent;

FIG. 3 is a photographic reproduction of a polyacrylamide gelelectrophoresis analysis of various 35-, 51- and 101-mers comprisingvarious percentages of dC^(Ac) and dC^(bz) and subjected to thecleavage/deprotection reagents of FIG. 2;

FIG. 4 is an electropherogram of a heterogeneous 51-mer comprising 35%dC^(bz) subjected to ammonia as a cleavage/deprotecting reagent;

FIG. 5 is an electropherogram of a heterogeneous 51-mer comprising 35%dC^(Ac) subjected to methylamine/t-butylamine as a cleavage/deprotectionreagent;

FIG. 6 is a photographic reproduction of PCR-derived 957 base-pairamplified template;

FIG. 7 is a photographic reproduction of a sequencing reaction of anM13mp18 template;

FIG. 8 is an electropherogram of a 3'-Terminal Transferase extensioninitiated using a 22-mer comprising dC^(Ac) and subjected tomethylamine/t-butylamine as a cleaving/deprotecting reagent; and

FIG. 9 is an electropherogram of a 3'-Terminal Transferase extensioninitiated using a 22-mer comprising dC^(bz) and subjected to ammonia asa cleaving/deprotecting reagent.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As those in the art appreciate, a drawback associated with automatedsynthesizers is the time necessary for deprotection of the protectinggroups and, if synthesized on a solid support material, cleavagetherefrom. Additionally, and from a practical perspective, for themajority of commercially available automated synthesizers that conductcleavage and deprotection on the instrument itself, that instrumentcannot be utilized during the cleavage step, which of course reducesthroughput. Additionally, conventional cleaving and deprotecting agentscan require an individual to manually push/pull ammonia through thesynthesizer column to effectuate cleavage and deprotection, thusreducing the time that the individual could be engaged in otherprojects. Furthermore, heating of the contents of the synthesizer columnwith ammonia typically requires pouring the insolubilizedoligonucleotide into a different container, adding ammonia, heating thenew container, and centrifuging the container to remove the supportmaterial. All of these steps are inherently time consuming and add thepotential for, inter alia, errors.

While a reagent which can reduce the time for deprotection and cleavageis of course an obvious goal, those in the art will appreciate that anysuch reagent must satisfy at least the following criteria: the integrityof the oligonucleotide must be preserved, i.e. the oligonucleotide mustbe biologically useful for e.g., use as probes, sequencing andsynthesizing, etc.; and, side product formation must be substantiallyminimized, and, preferably, excluded altogether.

As used herein, the term "oligonucleotide" is meant to encompasssynthetic deoxy- and ribo-oligonucleotides as well as modifiedoligonucleotides, i.e. where the 3'OH, 5'OH, sugar, or heterocyclic baseare modified, as well as modification of the phosphate backbone (e.g.methyl phosphonates, phosphorothioates, and phosphoramidates).Additionally, oligonucleotides can also include oligonucleotidescomprising an attached reporter group, e.g. biotin, avidin, haptens,dyes, fluorescent, chemiluminescent, enzymatic or radioactive labels,and solid supports other than the solid support from which theoligonucleotide is synthesized.

As used herein, the terms "rapid", "substantially shortened" "fast",etc. when used in relation to the disclosed reagent, are relative, anddepend upon the temperature of the medium in which the reagent isoperating. Broadly, these terms are intended to indicate thatdeprotection and cleavage can be completed within at least about 90minutes at room temperature, and within at least about 15 minutes atabout 90° C. Comparatively, ammonia requires at least about 24 hours atroom temperature for cleavage and deprotection, and at least about 30minutes at about 80° C.

It has been discovered that combinations of at least one first agentwhich is at least 5 times more nucleophilic than ammonia; at least onesecond agent that is at least 1.5 times less polar than water; andaqueous ammonia, can be effectively utilized as deprotection andcleavage reagents which substantially prevent transamination/sideproduct formation. "Combinations", as used herein in reference to thefirst agent, the second agent, and aqueous ammonia, is meant to indicatethat the reagent comprises at least the following preferred embodiments:

a) first agent;

b) first agent and aqueous ammonia;

c) first agent and second agent; and

d) first agent; aqueous ammonia; and second agent.

The first agent (more nucleophilic than ammonia) is a straight chainalkylamine having from between 1 to about 10 carbon atoms. In additionto the nucleophilicity thereof, the length of the alkylamine is also aconsideration, such that preferably, the straight chain alkylamine hasfrom between 1 to about 6 carbon atoms, more preferably from between 1to about 3 carbon atoms, and most preferably 1 carbon atom (i.e.,methylamine). Methylamine is a particularly preferred first agentbecause it is about 40 times less nucleophilic than ammonia and becauseit is the "smallest" of the defined straight chain alkylamines. Whilenot wishing to be bound to any particular theory, it is believed thatsuch agents, due to their nucleophilicity and size, are particularlywell suited to attack the bonds between the protecting groups (and thesolid support, if utilized) and the individual nucleotides of theoligonucleotide. Because of the relative "ease" at which such agents canattack these bonds (i.e., increase in reaction kinetics), the time fordeprotection and cleavage is substantially shortened.

As those in the art appreciate, "transamination" refers to the exchangeof amines on a nucleotide; typically, transamination is manifested asside-product formation. In the presence of, e.g., methylamine, thepotential for transamination of, e.g., cytidine to N-methylcytidine, anunwanted side-product, increases. Accordingly, it is preferred that thereagent comprises at least one second agent (less polar than water) as atransamination suppression agent (for convenience, hereinafter referredto as "TSA"). The TSA is preferably selected from the group consistingof straight-chain, branched, cyclic, saturated and unsaturatedalkylamines having from between 1 and about 10 carbon atoms and whichmay further comprise functional groups; ethanol; methanol;isopropylamine; acetylnitrile, dimethylformamide; tetrahydrofuran; andcombinations of the foregoing. Exemplary alkylamines as defined include,but are not limited to, t-butylamine, ethylamine, propylamine,isopropylamine, dimethylamine, diethylamine, trimethylamine andsecondary butylamine. The preferred TSAs have a polarity index valuethat is at least about 1.5 times less than that for water (the polarityindex value for water is about 10). While not wishing to be bound by anyparticular theory, it is believed that this feature of the TSAsubstantially reduces or prevents transamination from occurring, due tobeneficial interactions between the TSA with the nucleotide amines oragents comprising amines involved in a transamination event.

Aqueous ammonia can be added to the reagent, although this is not arequirement. The presence or absence of aqueous ammonia in the reagentis principally discretionary with the investigator. In essence, aqueousammonia does not significantly affect the reagent in terms of thecleavage and deprotection reactions; however, the presence of aqueousammonia in combination with the first agent can increase the timenecessary for deprotection by about 50%. Accordingly, although thereagent can comprise aqueous ammonia, preferably, the reagent does notinclude ammonia.

Preferred volume-to-volume ratios of the components of the reagent areas follows: for an embodiment of the reagent comprising a first agentand a TSA second agent, a preferred ratio is between about 9:1 to about1:9, more preferably from between about 7:3 to about 3:7, and mostpreferably about 1:1; for an embodiment of the reagent comprising afirst agent and aqueous ammonia, a preferred ratio is between about 9:1to about 1:9, more preferably from between about 7:3 to about 3:7, andmost preferably about 1:1; and for an embodiment of the reagentcomprising the first agent, a TSA second agent, and aqueous ammonia, apreferred ratio for the three components is from between about9-1:9-1:9-1 to about 1-9:1-9:1-9, more preferably from between about7-3:7-3:7-3 to about 3-7:3-7:3-7, and most preferably about 1:1:1.

The reaction temperature is principally dependent upon the needs of theinvestigator. I.e., the reaction temperature can range from betweenabout 25° C. to about 100° C. (although higher and lower temperaturescan be utilized as long as the integrity of the oligonucleotide is notsignificantly impacted whereby the resulting oligonucleotide hassubstantially limited utility). As those in the art appreciate,increasing the temperature of a chemical reaction typically increasesthe reaction kinetics thereof. Thus, when speed is a consideration (suchas in a commercial setting where it is desirable that oligonucleotidethroughput is high), it is preferred that the reaction temperature beabove room temperature, preferably greater than about 50° C., and mostpreferably between about 65° and about 80° C. While such temperatures donot have a substantial impact upon the cleavage time, such temperaturescan substantially decrease the time necessary for deprotection. However,it is to be understood that the reaction temperature can be at roomtemperature such that cleavage and deprotection is accomplished in lessthan about 90 minutes.

In accordance with the disclosure, a particularly preferred embodimentof the reagent comprises a 1:1 volume to volume ratio of methylamine andt-butylamine. A particularly preferred reaction temperature is about 65°C.; at this temperature, deprotection and cleavage of protected,insolubilized oligonucleotides can be accomplished in less than about 10minutes.

EXAMPLES

The following Examples, directed to preferred embodiments of theinvention are not intended, nor should they be construed to be,limitations on the disclosure or the claims to follow:

I. Materials and Methods A. Reagents 1. Cleavage/Deprotection Reagent

All chemicals were at least of ACS grade. Ammonium hydroxide wasobtained from Aldrich (Milwaukee, Wis.; Cat. No. 22, 122-8).Methylamine, 40 wt % solution in water, was obtained from Aldrich (Cat.No. M2,775-1), as was t-butylamine (Cat. No. B8, 920-5).

Methylamine/t-butylamine reagent was prepared by mixing a 1:1volume-to-volume ratio, followed by shaking for 5 minutes at roomtemperature and storage at 4° C. Ammonium hydroxide was stored inaccordance with supplier instructions.

2. Protected Deoxynucleosides

The following protected deoxynucleosides were obtained from SigmaChemical Co. (St. Louis, Mo.):

a) dA^(bz) (Cat. No. B 6130);

b) dC^(bz) (Cat. No. B 5882);

c) dC^(ibu) (Cat. No. I 6261); and

d) dG^(ibu) (Cat. No. I 6007).

Thymidine was obtained from Sigma (Cat. No. T 5018).

A novel protected deoxycytidine was also utilized for the Examples. Itwas determined that dC^(bz) and dC^(ibu) (the so-called "traditional"protected deoxycytidines), when used in conjunction with the disclosedreagent, could lead to side-product formation. The novel protecteddeoxycytidine comprises an acetyl ("Ac") protecting group. Synthesisthereof is disclosed in the co-pending application referenced above,which is incorporated fully herein by reference.

B. Commercially Available Protocols 1. Polymerase Chain Reaction ("PCR")

PCR analysis of oligonucleotide primers subjected to the disclosedcleavage/deprotection reagent was conducted using a Perkin Elmer CetusGeneAmp™ DNA Amplification Reagent Kit with AmpliTag™ (Part NO.N801-0055). Manufacturer instructions were followed.

2. DNA Sequencing

Sequencing reaction was performed using M13mp18 single stranded DNAtemplate (New England Biolabs, Cat. No. 404-C) following the protocol ofUnited States Biochemical Sequenase® Version 1.0, using α-[³⁵ S]-dATP.

C. Instruments 1. Automated DNA Synthesizer

Synthesis of oligonucleotides was performed using a Biosearch 8750™ DNAsynthesizer; controlled pore glass (CPG), 500Å-1000Å pore size, was usedfor the solid support material. Homo- and hetero-oligonucleotides ofvarious lengths were synthesized in accordance with manufacturerinstructions.

2. Capillary Electrophoresis

Capillary electrophoresis of oligonucleotides was performed on a BeckmanInstruments, Inc. P/ACE™ 2000 high performance capillary electrophoresissystem. A 37 cm U100P Urea Gel Column (Beckman, Cat. No. 338480) wasutilized. Samples were loaded onto the columns via the electrokineticinjection method (10 kV, 3 seconds); separation was conducted at 11kV/cm for 30-90 minutes, depending on oligonucleotide length.Tris-hydroxymethyl aminomethane ("TRIS")-borate 7M urea, running buffer(Beckman, Gel Buffer Kit, Cat. No. 338481) was utilized. Absorbancedetection was in the range of from 0.05 to 2.0 OD₂₆₀ nm /ml, dependingprincipally on the length of the oligonucleotide.

3. High Pressure Liquid Chromatography ("HPLC")

HPLC analysis was conducted on a Beckman Instruments System Gold™ HPLCProgrammable Solvent Module 126 equipped with a diode array detectormodule 168 and autosampler 507. A C₁₈ Ultrasphere™ HPLC column (Beckman,Cat. No. 235329; 5 μ particles, 4.6mm×25cm) was utilized. Bottle Acontained 0.1M ammonium acetate, pH 6.9; Bottle B contained HPLC-gradeacetonitrile. The system was operated in a gradient mode as follows (1ml/min. flow rate): 0-10 min: 85% Bottle A, 15% Bottle B; 20-25 min: 75%Bottle A, 25% Bottle B; 25-27 min: 50% Bottle A, 50% Bottle B; 27-30min: 50% Bottle A, 50% Bottle B; 30-35 min, 100% Bottle A, 0% Bottle B.

II. Example I. Cleavage of Support-Bound, Protected Oligonucleotides

Heterogeneous 21-mers were synthesized using the Biosearch 8750synthesizer. Four different types of 21mers were analyzed, whereby eachof the four nucleosides were tethered to the support, (i.e., the site ofcleavage); the remaining 20 nucleosides were identical. Accordingly, thedisclosed reagent was evaluated for cleavage time for each of the fournucleosides.

For comparative purposes, the following conditions were analyzed.

a) methylamine/t-butylamine (1:1, v/v);

b) methylamine; and

d) ammonia.

Support bound oligonucleotides were treated with 1 ml of the foregoingreagents by passing the reagents back-and-forth through the columncomprising the oligonucleotides via syringes placed on either end of thecolumn, at room temperature. Aliquots (50 μl) of the resulting mixtureswere removed at 1, 2, 5, 10, 15, 30 and 60 minutes, admixed with 1 mldouble-distilled H₂ O and absorbance was measured at 260nm.

Irrespective of the nucleoside immediately adjacent to the solidsupport, cleavage was accomplished in about 5 minutes for reagents (a)and (b) above. For ammonia, cleavage was accomplished in about 60minutes for all conditions. FIG. 1 provides a kinetics plot summarizingthe preceding, as a percentage of cleavage of the oligonucleotides overtime.

Example II: Deprotection of Protected Oligonucleotides

Deprotection of protected oligonucleotides was determined byreverse-phase HPLC analysis of enzymatically digested oligonucleotides.Enzymes utilized were: (1) phosphodiesterase I (Sigma, Cat. No. P-689F)reconstituted with 5 ml of 40 mM TRIS, and 10 mM MgCl₂, pH 7.5 (0.01 Uwas utilized per assay); and, (2) alkaline phosphatase (Sigma, Cat. No.P-4252) from E. coli 200 U/1.8 ml (0.2 U was utilized per assay). 25 μlof phosphodiesterase and 2 μl of phosphatase were added to 1-2OD_(260nm) of oligonucleotide, followed by incubation at roomtemperature for 30 minutes to 120 minutes. 0.5 to 1.0 OD_(260nm) of thedigested mixture was injected onto the above-referenced HPLC column.

Because deprotection of protected deoxyguanosine is the rate limitingstep, analysis of several 21-mer oligonucleotides consisting ofdifferent percentages of dG^(ibu) were investigated. Synthesis wasconducted using the above-referenced DNA synthesizer to produce thefollowing 21-mers:

1) 5'-CTG-GAC-AGT-AGT-CAG-ACT-GC-(T)-3' [25% G]

2) 5'-GTG-GAC-GCT-AGG-GCG-GAG-(C-(T)-3' [50% G]

3) 5'-Gx21-3' [100% G]

The tested reagents were the same as presented in Example I. Reactiontemperatures ranged from 25° to 80° C. Results are as follows in TableI:

                  TABLE I                                                         ______________________________________                                        Deprotection of Protected Oligonucleotides (time, in minutes)                 ______________________________________                                        Tem-   Methylamine/     Methylamine/                                          perature                                                                             t-butylamine     Ammonia                                               (°C.)                                                                         25% G   50% G   100% G 25% G 50% G 100% G                              ______________________________________                                        25     75      90      90     75    75    100                                 37     20      30      30     20    30    30                                  55     7       10      10     10    10    10                                  65     5        5       7     10     7     5                                  80     2        3       3      2     2     2                                  ______________________________________                                        Tem-                                                                          perature                                                                             Methylamine      Ammonia                                               (°C).                                                                         25% G   50% G   100% G 25% G 50% G 100% G                              ______________________________________                                        25     60      60      60     4320  4320  5940                                37     20      20      20     1200  1200  1800                                55     5       7       7       240   240   360                                65     5       5       5       180   180   240                                80     2       2       2       60    60    90                                 ______________________________________                                    

The foregoing data illustrates a variety of features of the disclosedreagent. Initially, it is noted that relative to ammonia, the so-called"traditional" cleavage/deprotection reagent, the disclosed reagentperforms both cleavage and deprotection in significantly shorter times.Additionally, it is noted that as temperature increases, the reactionkinetics also increase, as evidenced by the decrease in deprotectingtimes. Thus, it is possible to utilize the disclosed reagent across avariety of reaction temperatures, depending upon the needs of theinvestigator. Furthermore, it is noted that in terms of cleavage anddeprotection, not only do various embodiments of the disclosed reagentfunction on an approximate equivalent par, the various embodimentsperform significantly better than the traditional cleavage/deprotectionreagent.

Example III: Cytidine Side Product Formation

As noted, deoxycytidine is ordinarily most susceptible to side productformation during, e.g., deprotection of oligonucleotides comprisingdeoxycytidine. Typically, such side product formation is viatransamination during the deprotection step or after deprotection whenthe reagent continues to be in contact with the deprotecteddeoxycytidine.

As those in the art appreciate, the synthesis of oligonucleotides istypically conducted with the intent of retrieving the end-product asquickly as possible. Occasionally, however, it is possible that thesolubilized, protected oligonucleotide may remain in a deprotectionreagent for extended time periods. As those in the art furtherappreciate, such an increase in time when the oligonucleotide is withinthe reagent can increase the chance of a transamination event, thusincreasing the chance of side product formation.

Deoxycytidine side product formation was investigated by reverse phaseHPLC, using both methylamine and methylamine/t-butylamine as thereagent, across several times and temperatures. The "traditional"deoxycytidine protecting groups, "bz" and "ibu" were studied, as well asthe above-referenced "Ac" protecting group. The side product observedwhen utilizing such reagent (as confirmed by Nuclear Magnetic Resonance)was N-methyl cytidine. Percentage of N-methyldeoxycytidine formation,relative to deoxycytidine, are presented below, based uponsolution-based deprotection of deoxycytidine protected with an Acprotecting group:

                  TABLE II                                                        ______________________________________                                        Percentage of N-methylcytidine Formation*                                               TEMPERATURE                                                         Reagent     25° C.                                                                             37° C.                                                                          65° C.                                ______________________________________                                        Methylamine <0.01**     <0.01    <0.01                                                    (60 min.)   (20 min.)                                                                              (5 min.)                                     Methylamine ˜0.05 ˜0.25                                                                            ˜2.5                                   (16 hrs.)                                                                     Methylamine/                                                                              <0.01       <0.01    ˜0.6%                                  t-butylamine                                                                  (16 hrs.)                                                                     ______________________________________                                         *average percentages                                                          **0.01% is the lowest detectable limit of the instrument                 

These results indicate that for a typical oligonucleotide synthesis(i.e. one in which the investigator is desirous of obtaining thefinished end product as soon as possible), a reagent comprisingmehtylamine does not lead to statistically significant cytidine sideproduct formation. However, as the time that the oligonucleotide remainsin the reagent increases, so too does the formation of cytidine sideproduct formation. Thus, the use of a Transamination SuppressionReagent, (as defined) is useful; the data indicates that relative tomethylamine, a reagent comprising methylamine and the TSA t-butylaminesignificantly reduces cytidine side product formation.

Beneficially, then, a TSA can be included in the reagent whether or notsuch extended reaction times are contemplated. As should be appreciated,the use of a TSA, although ordinarily not ,necessary, is preferred forat least two reasons: first, the presence of the TSA reduces thepotential for side product formation; and second, the investigator isprovided with the opportunity to maintain the oligonucleotide within thereagent for periods in excess of optimal cleavage/deprotection times, ifsuch extended time periods are desired.

A secondary set of studies was conducted along these lines. For thesestudies, side product formation for dC^(Ac), dC^(ibu), dC^(bz),dG^(ibu), dA^(bz) and dT (as a relative percentage of non-side productformation for the nucleosides) were investigated at various times andtemperatures using methylamine/t-butylamine as the reagent. Results areas presented in Table III:

                  TABLE III                                                       ______________________________________                                        Percentage of Side Product Formation*                                         Temp.  Reaction                                                               (°C.)                                                                         Time     C.sup.AC                                                                              C.sup.ibu                                                                          C.sup.bz                                                                            G.sup.ibu                                                                          A.sup.bz                                                                            T                               ______________________________________                                        25     90    min.   **    0.15 10.0  **   **    **                            25     16    hrs.   **    ***  ***   **   **    **                            37     30    min.   **    0.15 10.0  **   **    **                            37     5     hrs.   **    ***  ***   **   **    **                            37     16    hrs.   **    ***  ***   **   **    **                            65     5     min.   **    0.15 10.0  **   **    **                            65     1     hr.    **    ***  ***   **   **    **                            65     16    hrs.   0.6   ***  ***   **   **    **                            80     3     min.   **    0.15 10.0  **   **    **                            80     1     hr.    ***   ***  **    **   **    **                            ______________________________________                                         *Averages                                                                     **<0.01                                                                       ***not investigated due to high percentages at optimal                        temperature/reaction time                                                

These results indicate at least several things. First, with respect tothe dC protection groups, the data indicates that an "Ac" protectinggroup can beneficially be utilized in conjunction with the disclosedreagent; the "traditional" cytidine protecting groups resulted insignificantly higher side product formation. Second, the disclosedreagent does not lead to statistically significant side productformation for any of the protected deoxynucleosides at any of theinvestigated temperatures or reaction times, with the exception ofdeprotection of dC^(Ac) at the elevated temperatures and at timesgreater than the desired reaction times. Thus, for oligonucleotidescomprising deoxycytidine protected with an Ac protecting group, it ispreferred that at such elevated temperatures, extended reaction timesnot be utilized.

Example IV: Enzymatic Digestion Analysis of Non-PurifiedOligonucleotides

Analysis of the composition of several oligonucleotides were conductedusing enzymatic digestion and reverse phase HPLC techniques. Thesestudies were conducted using deoxycytidines protected with an Acprotecting group and a traditional protecting group, bz; all otherprotecting groups were consistent between the oligonucleotides. 35-mers,51-mers and 101-mers, having the following sequences, where analyzed:##STR2## The oligonucleotides were cleaved and deprotected using anembodiment of the reagent comprising methylamine/t-butylamine at 25° C.for 90 minutes or ammonia for 3 hrs. at 65° C.; solubilized, deprotectedoligonucleotides were not purified prior to analysis. Results are asfollows in Table IV:

                  TABLE IV                                                        ______________________________________                                        Composition Analysis                                                                       Determined                                                                      Oligonucleotides                                                                           Oligonucleotides                                         Theoretical                                                                           Comprising dC.sup.Ao                                                                       Comprising dC.sup.bz                              ______________________________________                                        35-mer C     11        10.67      10.62                                              G      8         7.88       7.84                                              T      9         9.65       9.41                                              A      7         6.80       7.13                                       51-mer C     18        17.04      17.36                                              G     11        11.71      11.72                                              T     11        11.61      11.07                                              A     11        10.65      10.85                                       101-mer                                                                              C     35        33.80      33.53                                              G     22        20.74      20.70                                              T     22        21.78      21.73                                              A     22        25.09      25.04                                       ______________________________________                                    

The theoretical composition of the various non-purified oligonucleotidesand the determined composition provide good correlation. Additionally,the difference in deoxycytidine protecting groups, based upon the abovedata, does not indicate a statistically significant difference inresults.

Example V. Melting Point Determination

Melting point determinations were derived for a heterogeneous 21-mer andthe complement thereof; identical oligonucleotides, cleaved anddeprotected with either the disclosed reagent comprisingmethylamine/t-butylamine, or ammonia, when investigated. The 21-mer, andthe complement thereof, had the following sequences:

21-mer

5'-AGC-TAC-GGT-CAT-CGT-ATG-CAT-3'

Complement

5'-ATG-CAT-ACG-ATG-ACC-GTA-GCT-3'

Oligonucleotides were subjected to an embodiment of the reagentcomprising methylamine/t-butylamine, for 90 min. at 25° C., or ammoniafor 3 hrs. at 65° C. After deprotection and cleavage, 0.5 OD_(260nm) (in20 μl minimum volume) of each oligonucleotide and its complement wereadded to 1 ml of 10 mM TRIS, pH 7.5. Samples were boiled for 10-15minutes, followed by slow heating on a lead heating block. Samples wereplaced in a cuvette, and absorbance (260 nm) was followed from 25° C. to70° C. by raising the cuvette temperature at 3° C. intervals, followedby 3 minutes for stabilization and absorbance reading. Results arepresented in FIG. 2, where "." are the readings for the ammonia-treatedoligonucleotides, and "*" are the readings for the disclosed reagenttreated oligonucleotides.

The results presented in FIG. 2 indicate that the disclosed reagent,vis-a-vis ammonia, did not affect the melting point temperature of the21-mer. Absorbance readings versus temperature followed a nearlyidentical pattern under both reagent conditions.

Example VI. Polyacrylamide Gel Electrophoresis ("PAGE")

Analysis of 35-mers (35% dC^(bz) ; 35% dC^(Ac) ; 100% dC^(bz) ; and 100%dC^(Ac)), 51-mers (35% dC^(bz) ; 35% dC^(Ac) ; 100% dC^(bz) ; and 100%dC^(Ac)); and 101-mers (35% dC^(bz) ; 35% dC^(Ac) ; 100% dC^(bz) ; and100% dC^(Ac)) were analyzed by PAGE. The hetero 35-, 51- and 101-merswere as described in Example IV and for the homo 35-, 51- and 101-mers,the oligomer was synthesized from an insolubilized thymidine.Oligonucleotides comprising dC^(Ac) were cleaved and deprotected using areagent comprising methylamine/t-butylamine for 90 min. at 25° C.;oligonucleotides comprising dC^(bz) where cleaved and deprotected usingammonia for 3 hrs. at 65° C.

A 22 cm×16.5 cm denaturing gel was prepared by adding 107.3 ml ofdeionized water to 100 gm of premixed acrylamide/methylenebis-acrylamide (29:1) (Boehringer Mannheim Biochemicals, Indianapolis,Ind.; Cat. No. 100-151) to achieve a 50% stock solution. To 20 ml of the50% stock solution was added 22.5 g urea, 5 ml of 10×Tris-Borate/EDTA("TBE") and sufficient deionized water to achieve 50 ml. The solutionwas stirred and heated such that the solid constituents were dissolved.Thereafter, 20 mg ammonium persulfate and 20 μl N,N,N',N'-Tetramethylethylene diamine ("TEMD") was added; this solution was poured into cleanplates and allowed to polymerize for 1 hr. gels when pre-run with 1×TBEat 20 mA for 1 hr. 0.2-1.0 OD_(260nm) of each oligonucleotide was addedto 10 μl of 10 m urea. The 20 μl admixtures were loaded onto the gel andelectrophoresed at 28 mA for 2-4 hours, depending on the length of theoligonucleotide. Bands were visualized by UV shadowing on TLCfluorescent plate or by ethidium bromide staining.

Photographic results are presented in FIG. 3, where the lanes aredefined as follows:

    ______________________________________                                        Lane            Oligonucleotide                                               ______________________________________                                        1               35-mer (35% dC.sup.Ac)                                        2               35-mer (35% dC.sup.bz)                                        3               35-mer (100% dC.sup.Ac)                                       4               35-mer (100% dC.sup.bz)                                       5               51-mer (35% dC.sup.Ac)                                        6               51-mer (35% dC.sup.bz)                                        7               51-mer (100% dC.sup.Ac)                                       8               51-mer (100% dC.sup.bz)                                       9               101-mer (35% dC.sup.Ac)                                       10              101-mer (35% dC.sup.bz)                                       11              101-mer (100% dC.sup.Ac)                                      12              101-mer (100% dC.sup.bz)                                      ______________________________________                                    

The results of FIG. 3 indicate that the oligonucleotides subjected to anembodiment of the disclosed reagent and the Ac protection group providednearly identical PAGE patterns compared to the oligonucleotidessubjected to ammonia and the traditional deoxycytidine protecting group,bz.

Example VII. Capillary Electrophoresis

Heterogeneous 51-mer oligonucleotides comprising either 35% dC^(bz) or35% dC^(Ac) were subjected to either ammonia for 3 hrs. at 65° C. ormethylamine/t-butylamine for 90 min at 25° C., respectively, and wereanalyzed by capillary electrophoretic techniques. Electropherograms forthe oligonucleotide subjected to ammonia and an embodiment of thedisclosed reagent, are presented in FIG. 4 and 5, respectively.

The results of FIGS. 4 and 5 are nearly identical in terms of time fromsample introduction to detection of the 51-mer. The percent-of-totalintegrated areas beneath the major peaks, 66.902 for FIG. 4 and 66.575for FIG. 5, are also nearly identical. These results further indicatethat the reagent and the Ac deoxycytidine protecting group providecomparatively identical soluble, deprotected oligonucleotides vis-a-visammonia and the traditional deoxycytidine protecting group, bz.

Example VIII. Polymerase Chain Reaction

The foregoing Examples evidences that the disclosed reagent can beutilized to rapidly and efficiently cleave and deprotectoligonucleotides. As those skilled in the art appreciate, however, it isnecessary to be able to utilize such oligonucleotides for a variety ofprocedures.

Oligonucleotides used as primers in a polymerase chain reaction wheregenerated and subjected to an embodiment of the disclosed reagentcomprising methylamine/t-butylamine (where the deoxycytidines wereprotected with Ac) for 90 min. at 25° C. The primers were as follows:

18-mer

5'-CGC-CAG-GGT-TTT-CCC-AGT-3'

22-mer

5'-TTC-TGG-CGT-ACC-GTT-CCT-GTC-T-3'

The template was M13mp18 RFI DNA (New England Biolabs, Cat. No. 400-18).Manufacturer instructions were followed using the GeneAmp Reagent kit.

Initial melting temperature was 95° C. for 7 min.; 25 cycles were run ona Perkin Elmer Cetus DNA Thermal Cycler with the following cycleprofile:

    ______________________________________                                                    Temp. (°C.)                                                                     Time (sec)                                               ______________________________________                                        Seq. #1       94         1                                                    Seq. #2       94         60                                                   Seq. #3       37         1                                                    Seq. #4       37         120                                                  Seq. #5       72         1                                                    Seq. #6       72         180                                                  ______________________________________                                    

The resulting 957 base-pair PCR product was electrophoresed on a 1%agarose gel in TRIS-Acetate/EDTA ("TAE") and stained with ethidiumbromide. Photographic results are presented in FIG. 6 where thedesignated lanes are as follows:

    ______________________________________                                        Lane 1   957 bp product                                                                              (primers derived                                                              using methylamine/t-                                                          butylamine reagent                                                            and acetyl                                                                    protecting group for                                                          deoxycytidine);                                        Lane 2   957 bp product                                                                              (primers derived                                                              using ammonia and bz                                                          protecting group for                                                          deoxycytidine);                                        Lane 3   Gel Marker    (Lambda DNA digested                                                          with Hind III, 2322                                                           and 2027 bp                                                                   markers); and                                          Lane 4   Gel Marker    (PBR322 DNA digested                                                          with Hinf I, 1632                                                             and 506 bp marker)                                     ______________________________________                                    

The results presented in FIG. 6 indicate that primers derived utilizingan embodiment of the disclosed reagent and deoxycytidine protectinggroup, Ac, led to the production of an amplified product substantiallyidentical to that derived from primers generated by ammonia cleavage anddeprotection and using a bz protecting group for deoxycytidine.

Example IX. DNA Sequencing

Two sets of 18-mers were synthesized using the deoxycytidine protectinggroup, Ac, and bz, and were subjected to an embodiment of the disclosedreagent comprising methylamine/t-butylamine for 90 min. at 25° C., andammonia for 3 hrs. at 65° C., respectively. The 18-mers had thefollowing sequence:

18-mer

5'-CGC-CAG-GGT-TTT-CCC-AGT-3'

Solubilized, deprotected oligomers were purified using Sep Pak (Waters,Part No. 5190) DNA purification kit. These purified oligomers were usedas primers for sequencing purposes. The template was M13mp18 singlestranded DNA (New England Biolabs, Cat. No. 404-C); sequencing wasaccomplished using the 18-mers in conjunction with the USB Sequenasematerials and protocols. Results are presented in FIG. 7.

As the results of FIG. 7 indicate, the sequencing band patterns aresubstantially identical using primers subjected to the disclosed reagentand Ac protecting group vis-a-vis primers derived via ammonia and bz.

Example X. 3' Terminal Transferase Extension

22-mers were synthesized using the deoxycytidine protecting group, Ac,and bz, and were subjected to an embodiment of the disclosed reagentcomprising methylamine/t-butylamine for 90 min. at 25° C., and ammoniafor 3 hrs. at 65° C., respectively. The 22-mers had the followingsequence:

22-mer

5'-TTC-TGG-CGT-ACC-GTT-CCT-GTC-T-3'

Solubilized, deprotected oligomers were purified using Sep Pak DNApurification kit. These purified oligomers were used as primers for 3'terminal transferase extension studies.

2.5 OD_(260nm) of each oligonucleotide was added to 150 μl of deionizedwater; 5 mg Thymidine triphosphate ("TTP"), Sigma, Cat. No. T8635); 5 μlterminal deoxynucleotidyl transferase, 15 U/μl (BRL, Cat. No. 8008SB)and 50 μl trailing buffer. The admixture was incubated overnight at 37°C. and the resulting material purified using a Sep Pak C₁₈ cartridge asfollows: the reaction mixture was diluted 1:2 in 0.5 m ammonium acetate,loaded onto the cartridge, followed by washing of the cartridge withdeionized water, and the product eluted with 60% methanol in deionizedwater. The products were analyzed by capillary electrophoresis;electropherogram results are presented in FIGS. 8 and 9.

The electropherograms of FIGS. 8 and 9 evidence that the primerscomprising cytidine protected with Ac and subjected to the reagent (FIG.8) and primers comprising cytidine protected with bz and subjected toammonia (FIG. 9) were both extended at the 3' ends thereof, and that theresulting products were substantially identical.

The foregoing data evidences that the disclosed cleavage/deprotectionreagent rapidly and effectively removes oligonucleotides from a solidsupport and removes the protecting groups therefrom. Additionally, thesolubilized, deprotected oligonucleotides subjected to such reagent are,inter alia, biologically useful. Furthermore, the disclosed reagentcomprising a TSA substantially reduces side product formation.

By varying the times and temperatures at which oligonucleotides aresubjected to the reagent, the disclosed reagent can be utilized in avariety of contexts vis-a-vis synthesis of oligonucleotides. E.g.,increase throughput on automated synthesizers; cleavage and deprotectionof insolubilized and protected oligonucleotides; cleavage ofinsolubilized, protected oligonucleotides (i.e. where it is desirable tomaintain protected oligonucleotides in solution); deprotection ofsoluble oligonucleotides (i.e. where the investigator has storedsoluble, protected oligonucleotides; and deprotection of insolubleoligonucleotides (i.e., where the investigator has stored protected,insoluble oligonucleotides).

While the foregoing has been described in considerable detail, it is tobe understood that the embodiments disclosed in the Detailed Descriptionand Examples are not to be construed as limiting to the disclosure orthe claims to follow. The invention is not limited to automated DNAsynthesizers. The invention is not limited to deoxyribonucleic acidoligonucleotides, but can also be utilized with ribonucleic acidoligonucleotides and other modified oligonucleotides such asoligonucleotide methyl phosphonates and phosphorothioates. The inventionis not limited to the specified protecting groups for any of thenucleosides, but can be utilized with a variety of protecting groups.Modifications and changes that are within the purview of those skilledin the art are intended to fall within the scope of the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTGGACAGTAGTCAGACTGCT21                                                       (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTGGACGCTAGGGCGGAGCCT21                                                       (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGGGGGGGGGGGGGGGGGGGG21                                                       (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CAGTGCAGCTCCTAGCAGCCTAGCGTACTAGTCTT 35                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAGTCCTAG TCACAGTCCAGTCGCTCAAGCGTCCAG36                                       TTGCACAGGTCACCT51                                                             (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCTGCCAGTTCGGTCATCCGATCCTCGGTCACGCAA36                                        CTGTCAACGGCACCTA CTCCTCGTAACGTAGGACAG72                                       TCCGATTCGCACGTGCAAAGCCCATTCAT101                                              (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGCTACGGTCATCGTATGCAT21                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGCATACGATGACCGTAGCT 21                                                      (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CGCCAGGGTTTTCCCAGT 18                                                         (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TTCTGGCGTACCGTTCCTGTCT22                                                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: No                                                       (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CGCCAGGGTTTTCCCAGT18                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TTCTGGCGTACCGTTCCTGTCT22                                                  

What is claimed is:
 1. A reagent for cleaving a support boundoligonucleotide and/or removing at least one protecting group from aprotected oligonucleotide, the reagent comprising a volume ratio ofabout 1:1 aqueous methylamine:aqueous ammonia said aqueous methylamineconsisting essentially of 40 wt % methylamine amine in water and saidaqueous ammonia consisting essentially of about 29 wt % ammonia inwater.
 2. A reagent for cleaving a support bound oligonucleotide and/orremoving at least one protecting group from a protected oligonucleotide,the reagent comprising a volume ratio of from about 3:7 aqueousmethylamine:aqueous ammonia to about 7:3 aqueous methylamine:aqueousammonia said aqueous methylamine consisting essentially of 40 wt %methylamine amine in water and said aqueous ammonia consistingessentially of about 29 wt % ammonia in water.